1. Field of the Invention
The field of the invention relates to high throughput discovery of proteins fluorescently labeled at a cysteine residue (probe) that undergo a change in fluorescence ratio at 2 wavelengths upon binding an unbound analyte and which probes are used to measure levels of unbound analytes, including unbound free fatty acids and other unbound metabolites.
2. Description of the Related Art
For purposes of the present disclosure, “analytes” are molecules whose molecular weight is approximately 2000 Da or less and unbound analytes are these molecules in aqueous solution. These include metabolites and physiologically important molecules that occur naturally in the course of human or animal physiology or pathophysiology, and drug molecules and their metabolic products and nutrient molecules and their metabolic products. Depending upon their solubility, a fraction of each analyte is present as monomers in aqueous solution (either charged or neutral). This fraction is referred to as the “unbound analyte” fraction and includes unbound metabolites (METu).
For purposes of the present disclosure, “fatty acids”, a particular type of metabolite, are non-esterified carboxylated alkyl chains of 1-30 carbons atoms which may exist as neutral (e.g. protonated, sodium or potassium salt) or ionic species, depending upon the pH and conditions of the aqueous media. “Free fatty acids (FFA)” are equivalent to fatty acids and both terms refer to the totality of FFA including those in aqueous solution as monomers plus those that are not in solution (for example bound to other macromolecules (proteins, membranes), cells or part of an aggregate of FFA (micelles, soaps and other more complex aggregates). FFA present as monomers in aqueous solution (either charged or neutral) are referred to as “unbound free fatty acids (FFAu)”.
For the purposes of the present disclosure, the term “lipid” is taken to have its usual and customary meaning and defines a chemical compound which is most soluble in an organic solvent but has some level of solubility in the aqueous phase (the fraction that is unbound). Accordingly, a “lipid-binding protein” includes any protein capable of binding a lipid as lipid is defined herein.
Levels of unbound molecules, such as for example lipids, hormones and metabolic products, can provide information diagnostic of health and disease when measured in appropriate human or animal fluids. It is increasingly apparent that determination of the unbound (a.k.a ‘aqueous phase’ or ‘free’) concentration of such molecules provides critical information about physiologic homeostasis. Many metabolites are hydrophobic molecules with low aqueous solubility and unbound concentrations that are much lower than their “total” concentration, where the bulk of the “total” may be bound to proteins or cells. In biological fluids the concentration of the unbound molecules is often regulated to maintain a relatively constant unbound concentration under normal physiologic conditions. This regulation occurs through the interaction of the molecules with a carrier protein such as for example, albumin. Thus most of the molecules are generally bound to albumin, or other carriers. However a small fraction of the molecules may dissociate (and rebind) from the albumin into the aqueous phase and these are the unbound molecules.
Intracellular lipid binding proteins (iLBP) are a family of low-molecular weight single chain polypeptides. There are four recognized subfamilies. Subfamily I contains proteins specific for vitamin A derivatives such as retinoic acid and retinol. Subfamily II contains proteins with specificities for bile acids, eiconsanoids, and heme. Subfamily III contains intestinal type fatty acid binding proteins (FABPs) and Subfamily IV contains all other types of fatty acid binding protein (Haunerland, et al. (2004) Progress in Lipid Research vol. 43: 328-349). The entire family is characterized by a common 3-dimensional fold. Ligand binding properties of the different subfamilies overlap considerably. The wild type proteins of subfamily I (Richieri et al (2000) Biochemistry 39:7197-7204) and subfamily II both bind fatty acids as well as their native ligands. Moreover, single amino acid substitutions are able to interconvert the ligand binding properties of proteins of subfamilies I and II (Jakoby et al (1993) Biochemistry 32:872-878).
U.S. Pat. No. 5,470,714 and U.S. Pat. No. 6,444,432, which are incorporated herein by reference, describe probes for the determination of unbound free fatty acids (FFAu). These probes were constructed using either native or mutant forms of proteins from the iLBP family. As discussed above, this family includes FABPs (Banaszak et al (1994) Adv. Protein Chem. 45:89-151; Bernlohr et al (1997) Ann. Rev. Nutrition, 17: 277-303). FABPs are intracellular proteins of approximately 15 kDa molecular weight and have a binding site that binds 1 or 2 FFA.
For the purposes of the present disclosure “probes” are iLBPs that are fluorescently labeled at a cysteine residue and that undergo a change in the ratio of a fluorescence index measured at different 2 wavelengths upon binding an analyte. A probe may also be an iLBP fluorescently labeled at a cysteine residue with one fluorophore and at a different, preferably lysine, residue with a different fluorophore so that if the fluorescence of only one of the fluorophores change upon binding an analyte the ratio of fluorescence indices at 2 wavelengths will be different. Such probes may be used to determine the aqueous concentration of specific unbound analytes including FFAu, METu and other lipophilic hormones, and drugs, which is otherwise difficult because of their poor solubility properties in aqueous solutions. A change in the ratio of the fluorescence response is essential for the accurate determination of the intracellular as well as extracellular concentrations of unbound analytes.
Unfortunately, despite the availability of protein structures and co-complex structures with ligands of interest, existing state of the art of molecular theory is not sufficient to design probes with any desired specificity and sensitivity de novo. Thus, extensive experimentation is typically required to find protein probes that not only bind with the desired specificity, but also produce the required change in signal ratio indicative of ligand binding. Improving specificity and signaling through a completely random mutational strategy is not practical even for a small protein such as an FABP because a) there are 20131 possible mutants for a 131 residue FABP, and b) screening even a single probe for its binding specificity to a range of METu and other ligand molecules requires extensive time for purification, reaction chemistry and probe fluorescence response characterization.
Thus methods are needed to rapidly generate and screen thousands of resulting mutant probes. Each mutant needs to be produced, and chemically reacted with a fluorescent group, in sufficient quantity to enable the measurement of its sensitivity and selectivity for many different ligands (aka unbound analytes). It is also critical that the probes be as free as possible of contaminating proteins, unreacted fluorophore, and any other compounds that might interfere with sensitive fluorescence measurements. The development of a rapid, automated method for measuring and comparing probe responses to ligand is also critical.
Previously, probes found by these methods to be most effective in producing a change in signal ratio indicative of ligand binding were labeled with acrylodan primarily at the lysine 27 position of rat intestinal FABP of SEQ ID NO:2 or of mutations of SEQ ID NO:2 or SEQ ID NO 4 (Huber et al Biochemistry (2006) 45:14263-14274 and U.S. Pat. No. 7,601,510, which is incorporated herein by reference). Although labeling at the lysine position with environmentally sensitive fluorophores such as acrylodan results in probes that produce a fluorescence ratio change that is sensitive to ligand binding, we have found that the fluorophore labeling stoichiometry, the heterogeneity of labeling and the stability of the fluorophore-protein chemical bond, is mutant dependent. For example, acrylodan labeling of SEQ ID NO:2 or SEQ ID NO:2 with an L72A substitution, produce probes that are virtually completely labeled at a single lysine position (Lys 27) (for example, FIG. 2A. However, acrylodan labeling of other SEQ ID NO:2 muteins (SEQ ID NO:4) can result in probes that are not completely labeled and/or are labeled at more than a single position (for example FIG. 3).
Incompletely labeled muteins yield “probes” that are mixtures of fluorescently labeled and unlabeled muteins. As described previously (Richieri et al J. Biol. Chem. (1992) 267:23495-23501) the ligand (analyte) binding affinity is generally different for the labeled and unlabeled mutein and is usually smaller for the fluorescently labeled mutein than the unlabeled mutein. Moreover, typical variation in lysine labeling reaction conditions may result in lot-to-lot differences in the fraction of labeled and unlabeled mutein. This is an issue because the unbound ligand or analyte concentration determined using a probe that is a mixture of labeled and unlabeled mutein may be dependent upon the fraction of unlabeled mutein. This can occur because a significant fraction of the carrier (for example albumin) bound ligands (analytes) bind to the unlabeled mutein fraction of the probe solution. This effect may be exacerbated in the event that only a small fraction of the mutein is fluorescently labeled and therefore larger amounts of probe are required to yield a sufficiently intense fluorescence signal. Targeting lysine residues for fluorescence labeling is also potentially problematic because most iLBPs possess several lysine residues as well as a free N terminal amino group that can be labeled. In the event that multiple sites are labeled, the dynamic range of the probe's response to analyte binding will be compromised and the response may not reveal a single isoemissive point.
Mutating the iLBP protein to contain a single cysteine and using conditions to fluorescently label only cysteines should produce a homogenously labeled protein. However, substituting cysteine for lysine at position 27 of SEQ ID NO:2 followed by reaction with acrylodan results in fluorescently labeled protein that does not generate a fluorescence ratio change upon ligand binding (for example, FIG. 1B or IANBDE in Kleinfeld U.S. Pat. No. 5,470,714). Also in Kleinfeld '714 it is found that labeling the I-FABP mutants Thr81Cys or Thr83Cys with acrylodan yields a fluorescent protein that is unresponsive to FFAu binding. Other studies of fluorescently labeled cysteine iLBPs yield either a complete lack of sensitivity to ligand binding as in rat liver FABP (Evans and Wilton, Mol. Cell. Biochem. (2004) 98:135-140) or reveal a lack of change in ratio as in the CRABP proteins (Donato and Noy Anal Biochem (2006) 137:249-256). Thus methods are needed to generate cysteine labeled probes that exhibit the ratio response of the lysine labeled probes (Huber et al Biochemistry (2006) 45:14263-14274 and U.S. Pat. No. 7,601,510, which is incorporated herein by reference).
Embodiments of the invention described here satisfy these needs by disclosing methods for discovering locations for cysteine substitutions that allow rapid a) generation of large numbers (a library) of cysteine labeled mutein probes and the b) screening and of these probes to discover probes with different specificities for sets of unbound analytes and that respond to analyte binding by a change in fluorescence ratio. An important aspect of this invention is that it allows the previous necessary and very time consuming step of characterization of ligand binding to the protein to be omitted; only the probe itself is characterized. This is important not only for the avoidance of the protein characterization step but also because the properties of the probe are often not predictable from the ligand-protein binding characteristics. For example, different proteins can have very similar binding affinities but the fluorescence response of their derivative probes can be quite different.
Additional embodiments of the invention described here are methods for reducing the potential for fluorescence labeling at multiple lysine sites by mutating sets of lysines to arginine, thereby leaving only one fluorophore reactive site. Also described are methods to generate probes that are labeled with 2 different fluorophores. Such probes respond to analyte binding with a change in the ratio of a fluorescence index measured at two different wavelengths. This allows probes that do not reveal a ratio fluorescence index change in response to analyte binding to be converted into a ratio probe by labeling with a second fluorophore that reveals no or significantly reduced response to the binding of the analyte.